Methods and compositions for regulating Fas-associated apoptosis

ABSTRACT

This invention provides a novel purified mammalian protein designated FADD having the ability to bind the cytoplasmic region or domain of the Fas receptor. Also provided are nucleic acid molecules that encode the mammalian protein which binds the intracellular domain of Fas as well as methods for using the proteins and nucleic acid molecules.

This application is a continuation of application Ser. No. 08/416,379,filed Apr. 3, 1995, now abandoned.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made in part during work supported by the U.S.government, including a grant from the National Institutes of Health(NIH) CA61348. The government may have certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to Fas-mediated cellular functions and methodsfor the regulation of Fas-mediated cellular functions in a population ofcells.

BACKGROUND OF THE INVENTION

Programmed cell death (PCD) is a physiologic process essential to thenormal development and homeostatic maintenance of multicellularorganisms (reviewed in Vaux et al. (1994) Cell 76:777-779 and Ellis etal. (1991) Ann. Rev. Cell Biol. 7:663-698). Apoptosis, often equatedwith PCD, refers to the morphologic alterations exhibited by “actively”dying cells which include cell shrinkage, membrane blebbing andchromatin condensation. (For a general review of apoptosis, see Tomei,L. D. and Cope, F. O. Apoptosis: The Molecular Basis of Cell Death(1991) Cold Spring Harbor Press, N.Y.; Tomei, L. D.; Cope, F. O.Apoptosis II: The Molecular Basis of Apoptosis in Disease (1994) ColdSpring Harbor Press, New York; Duvall and Wyllie (1986) Immun. Today7(4):115-119 and Cohen (1993) Immunol. Today 14:126-130.) In contrast,necrosis, sometimes referred to as accidental cell death, is defined bythe swelling and lysis of cells that are exposed to toxic stimuli.

Apoptosis has been linked to many biological processes, includingembryogenesis, development of the immune system, elimination ofvirus-infected cells, and the maintenance of tissue homeostasis.Apoptosis also occurs as a result of human immunodeficiency virus (HIV)infection of CD4⁺ T lymphocytes (T cells). Indeed, one of the majorcharacteristics of AIDS is the gradual depletion of CD4⁺ T lymphocytesduring the development of the disease. Several mechanisms, includingapoptosis, have been suggested to be responsible for the CD4 depletion.It is speculated that apoptotic mechanisms might be mediated eitherdirectly or by the virus replication as a consequence of the HIVenvelope gene expression, or indirectly by priming uninfected cells toapoptosis when triggered by different agents.

The depletion of CD4⁺ T cells results in the impairment of the cellularimmune response. It has been reported that an inappropriateactivation-induced T cell PCD causes the functional and numericalabnormalities of T_(H) cells from HIV-infected patients, that leads tothe near collapse of the patient's immune system. (Brunner, T. et al.(1995) Nature 373:441-444; Dhein, J. et al. (1995) Nature 373:438-441;and Ju, S-T. et al. (1995) Nature 373:444-448).

Therefore, it is advantageous to block apoptosis and the ensuingdepletion of T cells, especially in HIV infected individuals.Accordingly, a need exists to maintain T cell function and viability inHIV infected individuals and to provide systems to screen for new drugsthat may assist in maintaining the cellular immune response. Thisinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

This invention provides a novel purified mammalian protein designatedFADD having the ability to bind the cytoplasmic region or domain of theFas receptor.

Also provided by this invention are nucleic acid molecules that encodethe mammalian protein which binds the intracellular domain of Fas.

An antibody, such as a monoclonal antibody, with specific affinity forFADD is further provided by this invention.

Methods of using the proteins, nucleic acids and antibodies describedabove are further provided herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that FADD specifically interacts with the cytoplasmicdomain of Fas in yeast. β-galactosidase filter assays performed on Y190yeast expressing the GAL4 activation domain-FADD fusion protein andindicated heterologous GAL4 DNA binding domain fusion proteins.

FIGS. 2A through 2D show sequence analysis of FADD and its novel deathdomain. FIGS. 2A through 2C (Seq. ID Nos. 1 and 2) is the cDNA sequenceof FADD and the deduced amino acid sequence of the FADD protein product.The boxed nucleotides represent an in-frame stop codon 130 base pairsupstream of the initiator methionine. The 5′ end of clones 8 and 15isolated in the yeast two-hybrid screen are indicated with arrows. Thedeath domain is underlined while the valine residue altered to anasparagine in FADDmt is indicated by the closed triangle. A potentialpoly (A) adenylation signal (ATTAAA) is overlined.

FIG. 2D (Seq. ID Nos. 3 through 6) shows the death domain of FADD andits amino acid sequence homology to other death domains. Solid blackshading refers to identical residues and gray shading indicatesconservative amino acid substitutions relative to the sequence of FADD.The arrow indicates the amino acid residue, which when substituted by anasparagine, disrupts binding and/or signaling in the respectiveproteins.

FIGS. 3A and 3B show that FADD is expressed in a variety of tissues anddevelopmental stages. In FIG. 3A, a human adult tissue Northern blot(Clontech) was probed with FADD cDNA, PBL=peripheral blood leukocyte.FIG. 3B is a human fetal Northern blot (Clontech) which was probed as inFIG. 3A.

FIGS. 4A through 4C show the specific interaction of GST-Fas andGST-Fas-FDS with in vitro translated FADD and FADD expressed intransfected 293T cells. FIG. 4A is a schematic representation of the GSTfusion proteins containing the cytoplasmic domains of Fas, Fas mutants,and TNFR-1. Amino acid residues are given for selected junctions andnumbering is based on the mature form of the receptor. The Lpr mutant(V²³⁸→N²³⁸) of Fas is represented by an asterisk. The gray shadingrepresents the death domain of FAS. Binding of FADD to the various GSTfusion proteins is depicted to the right.

FIG. 4B shows the interaction of in vitro translated, ³⁵S-labeled FADDwith various GST fusion proteins immobilized on glutathione-Sepharosebeads. After the beads were washed, retained FADD protein was analyzedby SD5-PAGE and autoradiography (upper panel). The gel was Coomassiestained and the bands representing the various GST fusion proteins werealigned to show equivalency of loading (lower panel).

In FIG. 4C, 293T cells were transfected with HA-epitope tagged FADD(HA-FADD) and metabolically labeled with ³⁵S-methionine and cysteine.Detergent lysates were prepared and incubated with the various GSTfusion proteins immobilized on glutathione-Sepharose beads. Afterwashing, the completed beads were dissociated and immunoprecipitatedwith an anti-HA (α-HA) antibody which should recognize HA-FADD. Thesamples were then analyzed by SDS-PAGE and autoradiography (upperpanel). The respective GST fusion proteins were shown as in B (lowerpanel).

FIGS. 5A through 5C show in vivo association of FADD with Fas andFas-FD5. FIG. 5A is a schematic representation of Fas and Fas mutantstransfected into 293T cells. The black square represents theFLAG-epitope tag engineered 5 amino acids downstream of the putativesignal sequence of Fas. The open rounded rectangles represent the 3cysteine-rich subdominals of the extracellular domain of Fas, while thecytoplasmic residues contain the death domain (gray rectangle) and aputative negative regulatory domain (shaded oval). Residue numbering isbased on the mature form of the receptor and the amino acid sequence isgiven for selected junctions. The Lpr mutant (V²³⁸→N²³⁸) of Fas isrepresented by an asterisk. In vivo FADD binding is described to theright of the schematic along with relative cell death caused by Fas andits mutants as described by Itoh et al. (1993) Cell 66:233-243.

For the results shown in FIG. 5B, 293T cells were cotransfected withHA-FADD and FLAG-epitope tagged Fas and Fas mutants (as depicted in FIG.5A) and metabolically labeled with 3S methionine and cysteine. Detergentlysates were then immunoprecipitated with anti-FLAG (α-FLAG) mAb andIsotype-matched control antibody and analyzed by SDS-PAGE andautoradiography to show expression of FLAG-tagged Fas and Fas mutants.White asterisks indicate relative position of Fas and its mutants.

In the results shown in FIG. 5C, 293T lysates (as in FIG. 5B) also wereimmunoprecipitated with α-HA antibody to show HA-FADD expression.

FIG. 5D shows the coimmunoprecipitation of FADD with Fas and mutants. Afraction of the α-HA immunoprecipitates (used in FIG. 5C) weredissociated and reimmunoprecipitated with an α-FLAG antibody.

FIG. 6 shows that FADDmt fails to bind Fas, suggesting a death domain todeath domain interaction. 293T cells were transfected with AU1-epitopetagged FADD (AU1-FADD) or AU1-FADDmt metabolically labeled with³⁵S-methionine and cysteine. Detergent lysates were prepared andincubated with various GST fusion proteins immobilized onglutathione-Sepharose beads. The samples were analyzed by SDS-PAGE andautoradiography (upper panel). The respective GST fusion proteins areshown as in FIG. 4B (middle panel). To show that equivalent amounts ofAU1-FADD and AU1-FADDmt were expressed and subsequently incubated withthe beads, an aliquot of the respective lysates was immunoprecipitatedwith α-AU1 antibody and visualized by SDS-PAGE and autoradiography(bottom panels).

FIGS. 7A through 7C show expression of FADD in BJAB cells inducesapoptosis which is inhibitable by CrmA. Shown in FIG. 7A is a previouslycharacterized BJAB cell line expressing CrmA (as described in Tewari etal. (1995) J. Biol. Chem. 270:3255-3260) and a corresponding vectorcontrol line that were transiently transfected with pCMV β-galactosidasein the presence or absence of an equimolar quantity of pcDNA3 AU1-FADD.The cells were cytocentrifuged, fixed, and stained for β-galactosidase(yellow) and with propidium iodide (red). Vector control linetransfected with β-galactosidase (Panel 1). Vector control linetransfected with β-galactosidase and pcDNA3-AU1-FADD (Panel 2).CrmA-expressing line transfected with β-galactosidase (Panel 3).CrmA-expressing line transfected with β-galactosidase andpcDNA3-AU1-FADD (Panel 4).

In FIG. 7B, at least 100 transfected cells, processed as in FIG. 7A,were counted and designated as apoptotic or non-apoptotic as determinedby cell morphology.

FIG. 7C shows immunostaining of AU1-FADD (green) which was transientlytransfected into a BJAB cell line expressing CrmA. Propidum iodidestaining (red) reveals nuclei.

FIG. 8 is a schematic model for the interaction between FADD and Fas.

DETAILED DESCRIPTION OF THE INVENTION

Although the morphologic features of cell death are well described, themolecular mechanisms behind apoptosis remain undefined. Recent work onPCD in the nematode Caenorhabditis elegans suggests that CED-3 initiatesthe cell death program (Yuan et al. (1993) Cell 75:641-652). Sequenceanalysis revealed that CED-3 is similar to the mammalian interleukin-1β(IL-1β) converting enzyme (ICE); a cysteine proteinase involved in theprocessing and activation of pro-IL-1β to the active cytokine (Cerrettiet al. (1992) Science 256:97-100 and Thornberry et al. (1992) Nature356:768-774). Overexpression of ICE in mammalian cells inducedapoptosis, suggesting that ICE, or a related protease, may be anessential component of the cell death pathway (Miura et al. (1993) Cell75:653-660).

If a CED-3 like protease is presumed to be a distal effector of themammalian cell death pathway, the proximal components that lead to itsactivation remained to be identified.

Two cell surface cytokine receptors, Fas/APO-1 antigen and the receptorfor Tumor Necrosis Factor (TNF), have been shown to trigger apoptosis bynatural ligands or specific agonist antibodies (Baglioni, C. (1992) TheMolecules and Their Emerging Roles in Medicine (Raven Press, N.Y.,N.Y.); Yonehara et al. (1989) S. J. Exp. Med. 169:1747-1756; Itoh et al.(1991) Cell 66:233-243; Trauth, B. C. et al. (1989) Science245:301-305). The Fas antigen is involved in the negative selection ofthymic T-lymphocytes and mice carrying a point mutation in thecytoplasmic domain of Fas exhibit a lupus-like lymphoproliferativeautoimmune disorder (Lpr, Watanabe-Fukunaga et al. (1992) Nature356:314-317. Recently, the Fas-mediated cell pathway has been implicatedin the activation-induced death of T-cells (Dhein et al., J. (1995)supra; Brunner, T. et al. (1995); supra; and Ju et al. (1995) supra.)While the main activity of Fas is to trigger cell death, the TNFreceptor (TNFR) can signal an array of diverse activities such asfibroblast proliferation, resistance to chlamidlae and synthesis ofprostaglandin E₂ (Tartaglia, L. A. et al. (1992) Immunol. Today13:151-153.

The activation of Fas and TNFR is caused by receptor aggregationmediated by the respective ligands or agonist antibodies. The signal isthought to be transduced by clustering of the intracellular domain(Boldin, M. P. et al. (1995) J. Biol. Chem. 270:387-391 and Song, H. Y.et al. (1994) J. Biol. Chem. 269:22492-22495) which encompasses a regionwhich is significantly conserved in the Fas antigen as well as in TNFR-1(Tartaglia et al. (1993) supra and Itoh, N. et al. (1993) J. Biol. Chem.266:10932-10937). This shared “death domain” suggests that bothreceptors interact with a related set of signal transduction moleculesthat had, until this disclosure, remained unidentified.

Provided herein is the molecular cloning and characterization of “FADD”a Fas Associating protein with) a novel Death Domain. The specificinteraction of Fas and FADD is due to the association of theirrespective homologous death domains. Remarkably similar to Fas-inducedkilling, overexpression of FADD induces apoptosis which is inhibitableby CrmA. Accordingly, FADD is a component of the Fas-receptor mediatedpathway and particularly Fas-induced apoptosis.

Proteins and Polypeptides

This invention provides purified proteins having the ability to bind thecytoplasmic region of the Fas receptor. In one embodiment, the purifiedproteins of this invention, termed “FADD proteins” are defined by theirspecific ability to bind to the cytoplasmic domain of the Fas receptorand Fas-FD5, a mutant of Fas possessing enhanced killing activity, butnot the functionally inactive mutants Fas-LPR and Fas-FD8.

In one embodiment of this invention, a purified protein is a 208 aminoacid human protein having an apparent molecular weight of about 22 to 24kDa and more particularly about 23.3 kDa, as determined by an SDSpolyacrylamide gel under reducing conditions. In a separate embodiment,a protein has the amino acid sequence shown in FIGS. 2A through 2C andSequence ID. No. 2). Also provided by this invention are polypeptidefragments of the mammalian protein, the human 23.3 kD protein or theprotein having the amino acid sequence shown in FIGS. 2A through 2C,each defined by the ability to bind to the cytoplasmic domain of the Fasreceptor using, for example, the in vitro binding assay described below.These polypeptide fragments can include any fragment containing fromabout amino acid 41 to amino acid 208 or the C-terminal half of FADD asdepicted in FIGS. 2A through 2C.

It is understood that functional equivalents of the protein also shownin FIGS. 2A through 2C, the 23.3 kD purified protein, or the polypeptidefragments thereof, e.g., as shown in FIGS. 2A through 2C, andequivalents thereof, also are within the scope of this invention. Onesuch equivalent includes chemical structures other than amino acidswhich functionally mimic the binding of FADD to the cytoplasmic domainof the Fas receptor (“muteins”). An additional example of an equivalentis a protein or polypeptide containing a distinct protein or polypeptidejoined to FADD or its equivalent which varies the primary sequence ofprotein of this invention from the sequences provided in FIGS. 2Athrough 2C without necessarily affecting the binding of the resultantpolypeptide or protein to the cytoplasmic domain of Fas. Where specificamino acids or other structures or sequences beyond the sequence shownin FIGS. 2A through 2C are presented, it is intended that variousmodifications which do not destroy the function of the binding site arewithin the definition of the proteins encompassed by this invention. Forthe purposes of this invention, the term “FADD protein” is intended tomean all of the proteins, polypeptides, fragments and equivalentsthereof, having the ability to bind the cytoplasmic domain of Fas.

An agent having the ability to inhibit the ability of FADD to bind tothe cytoplasmic domain of Fas receptor is further provided by thisinvention. Such agents include, but are not limited to, an anti-FADDantibody, a dominant inhibitory fragment of FADD or a solubleintracellular Fas. “Soluble intracellular Fas” is an intracellularportion of the Fas receptor which binds FADD.

The terms “proteins” and “polypeptides” also are intended to includemolecules containing amino acids linearly coupled through peptide bonds.As used herein, the term “peptide bond” or “peptide linkage” refers toan amide linkage between a carboxyl group of one amino acid and theα-amino group of another amino acid. Such polypeptides also can containamino acid derivatives or non-amino acid moieties. The amino acids canbe in the L or D form so long as the binding function of the polypeptideis maintained. The term amino acid refers both to the naturallyoccurring amino acids and their derivatives, such as TyrMe and PheCl, aswell as other moieties characterized by the presence of both anavailable carboxyl group and an amine group. Non-amino acid moietieswhich can be contained in such polypeptides include, for example, aminoacid mimicking structures. Mimicking structures are those structureswhich exhibit substantially the same spatial arrangement of functionalgroups as amino acids but do not necessarily have both the α-amino andα-carboxyl groups characteristic of amino acids.

As used herein, the term “hydrophobic” is intended to include thoseamino acids, amino acid derivatives, amino acid mimics and chemicalmoieties which are non-polar. Hydrophobic amino acids include Phe, Val,Trp, Ile and Leu. As used herein, the term “positively charged aminoacid” refers to those amino acids, amino acid derivatives, amino acidmimics and chemical moieties which are positively charged. Positivelycharged amino acids include, for example, Lys, Arg and His.

The proteins and polypeptides of this invention are distinct from nativeor naturally occurring proteins or polypeptides because they exist in apurified state. As used herein, the term “purified” when referring to aprotein or a polypeptide or any of the intended variations as describedherein shall mean that the compound or molecule is substantially free ofcontaminants normally associated with a native or natural environment.

The proteins and polypeptides of this invention can be obtained by anumber of methods well known to those of skill in the art, which includepurification, chemical synthesis and recombinant methods. For example,the proteins and polypeptides can be purified from a Fas₊ cell or tissuelysates using methods such as immuno-precipitation with anti-FADDantibody, and standard techniques such as gel filtration, ion-exchange,reversed-phase, and affinity chromatography using a FADD fusion proteinas shown herein. For such methodology, see for example Deutscher et al.,Guide to Protein Purification: Methods in Enzymology (1990) Vol. 182,Academic Press.

The proteins and polypeptides also can be obtained by chemical synthesisusing a commercially available automated peptide synthesizer such asthose manufactured by Applied Biosystems, Inc., Model 430A or 431A,Foster City, Calif. and the amino acid sequence provided in FIGS. 2Athrough 2C. The material so synthesized can be precipitated and furtherpurified, for example by high performance liquid chromatography (HPLC).

Alternatively, the proteins and polypeptides can be obtained bywell-known recombinant methods as described, for example, in Sambrook etal., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring HarborLaboratory (1989)) using the host vector systems described andexemplified below.

The FADD protein and polypeptides have several utilities. For example,they can be bound to a column and used for the purification of Fasreceptors. They also are useful as immunogens for the production ofanti-FADD antibodies as described below. They have further utility in anin vitro assay system to screen for agents or drugs which either inhibitor augment the FADD/Fas-receptor pathway and to test possible therapies.

When used to detect Fas or to screen for new Fas-regulating agents, FADDcan be bound to a solid phase carrier for example, glass, polystyrene,polyethylene, dextran, nylon, natural and modified celluloses,polyacrylamides, glutathione-agarose beads and agaroses. Those skilledin the art will know of other suitable carriers for this purpose.Accordingly, this invention also provides a method of detecting Fas in acell sample by first immobilizing FADD onto a solid support such asglutathione-agarose beads at a suitable concentration, eg., betweenabout 5 mg/ml to about 12 mg/ml, and more preferably between about 6mg/ml and about 10 mg/ml. The sample containing or suspected ofcontaining Fas is prepared and contacted with the beads under conditionsfavoring binding between the Fas receptor and FADD. Suitable conditionsare for example, those set forth in the experiment section describedbelow. The beads are then subjected to conditions to release the complexfrom the solid support and protein complex can then be visualized byautoradiography.

The proteins of this invention also can be combined with various liquidphase carriers, such as sterile or aqueous solutions, pharmaceuticallyacceptable carriers as defined below, suspensions and emulsions.Examples of non-aqueous solvents include propyl ethylene glycol,polyethylene glycol and vegetable oils. When used to prepare antibodies,the carriers also can include an adjuvant which is useful tonon-specifically augment a specific immune response. A skilled artisancan easily determine whether an adjuvant is required and select one.However, for the purpose of illustration only, suitable adjuvantsinclude, but are not limited to Freund's Complete and Incomplete,mineral salts and polynucleotides.

Nucleic Acids

Isolated nucleic acid molecules which encode amino acid sequencescorresponding to FADD protein, mutein, antibodies and active fragmentsthereof are further provided by this invention. As used herein, “nucleicacid” shall mean single and double stranded DNA, cDNA and RNA, includinganti-sense RNA. One can obtain an anti-sense RNA using the sequenceprovided in FIGS. 2A through 2C and the methodology described in VanderKrol et al. (1988) BioTechniques 6:958. “Isolated” means separated fromother cellular components normally associated with DNA or RNAintracellularly.

In one aspect of this invention, the nucleic acid molecule encoding FADDprotein or polypeptide has the sequence or parts thereof shown in FIGS.2A through 2C.

The invention also encompasses nucleic acid molecules which differ fromthat of the nucleic acid molecules shown in FIGS. 2A through 2C, butwhich produce the same phenotypic effect. These altered, butphenotypically equivalent nucleic acid molecules are referred to“equivalent nucleic acids.” Examples of such “equivalent nucleic acids”are those molecules which have a sequence which is homologous tosequence of in FIGS. 2A through 2C, and preferably have a homology ofgreater than about 50%, more preferably in excess of 90%. A homology ofabout 99% is most preferred. This invention also encompasses nucleicacid molecules characterized by changes in non-coding regions that donot alter the phenotype of the polypeptide produced therefrom whencompared to the nucleic acid molecule described hereinabove. Thisinvention further encompasses nucleic acid molecules which hybridize tothe nucleic acid molecule of the subject invention.

The nucleic acid molecules of this invention can be isolated using thetechnique described in the experimental section described below orreplicated using PCR (Perkin-Elmer) and the methods described below. Forexample, the sequence can be chemically replicated using PCR(Perkin-Elmer) which in combination with the synthesis ofoligonucleotides, allows easy reproduction of DNA sequences. The PCRtechnology is the subject matter of U.S. Pat. Nos. 4,683,195, 4,800,159,4,754,065, and 4,683,202. Alternatively, one of skill in the art can usethe sequence provided herein and a commercial DNA synthesizer toreplicate the DNA. RNA can be obtained by using the isolated DNA andinserting it into a suitable cell where it is transcribed into RNA. TheRNA can then be isolated using methods well known to those of skill inthe art, for example, as set forth in Sambrook et al. (1989) supra.

The invention further provides the isolated nucleic acid moleculeoperatively linked to a promoter of RNA transcription, as well as otherregulatory sequences for replication and/or expression of the DNA orRNA. As used herein, the term “operatively linked” means positioned insuch a manner that the promoter will direct the transcription of RNA offthe nucleic acid molecule. Examples of such promoters are SP6, T4 andT7. Vectors which contain a promoter or a promoter/enhancer, withtermination codons and selectable marker sequences, as well as a cloningsite into which an inserted piece of DNA can be operatively linked tothat promoter are well known in the art. See for example, Gacesa andRamji, Vectors: Essential Data Series (1994) John Wiley & Sons, N.Y.,which contains maps, functional properties, commercial suppliers and areference to GenEMBL accession numbers for various suitable vectors.Preferable, these vectors are capable of transcribing RNA in vitro or invivo.

Fragments of the sequence shown in FIGS. 2A through 2C, e.g., a fragmentcomprising nucleic acids coding for amino acid residues 111-170 as shownin FIGS. 2A through 2C, and its equivalents are useful as probes toidentify transcripts of the protein which may or may not be present. Thefragments also may be used as PCR primers. These nucleic acid fragmentscan by prepared, for example, by restriction enzyme digestion of thenucleic acid molecule of FIGS. 2A through 2C and then labeled with adetectable marker such as a radioisotope using well known methods.Alternatively, random fragments can be generated using nick translationof the molecule. For methodology for the preparation and labeling ofsuch fragments, see Sambrook et al., Molecular Cloning: A LaboratoryManual Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989) supra.Nucleic acid fragments of at least 10 nucleotides are useful ashybridization probes primers. Isolated nucleic acid fragments also areuseful to generate novel peptides. These peptides, in turn, are usefulas immunogens for the generation of polyclonal and monoclonalantibodies.

As noted above, an isolated nucleic acid molecule of this invention canbe operatively linked to a promoter of RNA transcription. These nucleicacid molecules are useful for the recombinant production of FADD andanti-FADD proteins and polypeptides or as vectors for use in genetherapy. Accordingly, this invention also provides a vector havinginserted therein an isolated nucleic acid molecule described above, forexample, a viral vector, such as bacteriophages, baculoviruses andretroviruses, or cosmids, plasmids and other recombination vectors.Nucleic acid molecules are inserted into vector genomes by methods wellknown in the art. For example, insert and vector DNA can both be exposedto a restriction enzyme to create complementary ends on both moleculesthat base pair with each other and which are then joined together with aligase. Alternatively, synthetic nucleic acid linkers can be ligated tothe insert DNA that correspond to a restriction site in the vector DNA,which is then digested with a restriction enzyme that recognizes aparticular nucleotide sequence. Additionally, an oligonucleotidecontaining a termination codon and an appropriate restriction site canbe ligated for insertion into a vector containing, for example, some orall of the following: a selectable marker gene, such as neomycin genefor selection of stable or transient transfectants in mammalian cells;enhancer/promoter sequences from the immediate early gene of humancytomegalovirus (CMV) for high levels of transcription; transcriptiontermination and RNA processing signals from SV40 for mRNA stability;SV40 polyoma origins of replication and ColE1 for proper episomalreplication; versatile multiple cloning sites; and T7 and SP6 RNApromoters for in vitro transcription of sense and anti-sense RNA.

An additional example of a vector construct of this invention is abacterial expression vector including a promoter such as the lacpromoter and for transcription initiation, the Shine-Dalgarno sequenceand the start codon AUG (Sambrook et al., (1989) supra). Similarly, aeucaryotic expression vector is a heterologous or homologous promoterfor RNA polymerase II, a downstream polyadenylation signal, the startcodon AUG, and a termination codon for detachment of the ribosome. Suchvectors can be obtained commercially or assembled by the sequencesdescribed in methods noted above.

Expression vectors containing these nucleic acids are useful to obtainhost vector systems to produce FADD and anti-FADD proteins andpolypeptides. It is implied that these expression vectors must bereplicable in the host organisms either as episomes or as an integralpart of the chromosomal DNA. Suitable expression vectors include viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, etc. Adenoviral vectors are particularly useful for introducinggenes into tissues in vivo because of their high levels of expressionand efficient transformation of cells both in vitro and in vivo. When anucleic acid is inserted into a suitable host cell, e.g., a procaryoticor a eucaryotic cell and the host cell replicates, the protein can berecombinantly produced. Suitable host cells will depend on the vectorand can include mammalian cells, animal cells, human cells, simiancells, insect cells, yeast cells, and bacterial cells constructed usingwell known methods. See Sambrook et al., (1989) supra. In addition tothe use of viral vector for insertion of exogenous nucleic acid intocells, the nucleic acid can be inserted into the host cell by methodswell known in the art such as transformation for bacterial cells;transfection using calcium phosphate precipitation for mammalian cells;or DEAE-dextran; electroporation; or microinjection. See Sambrook et al.(1989) supra for this methodology. Thus, this invention also provides ahost cell, e.g. a mammalian cell, a animal cell, a human cell, or abacterial cell, containing a nucleic acid molecule encoding a FADD oranti-FADD protein or polypeptide.

Using the host vector system described above, a method of producingrecombinant FADD or anti-FADD or active fragments thereof is provided bygrowing the host cells described herein under suitable conditions suchthat the nucleic acid encoding the FADD or anti-FADD protein orpolypeptide is expressed. Suitable conditions can be determined usingmethods well known to those of skill in the art, see for example,Sambrook et al., (1989) supra. Proteins and polypeptides purified fromthe cellular extract and thereby produced in this manner also areprovided by this invention.

A vector containing the isolated nucleic acid encoding FADD or anti-FADDprotein also is useful for gene therapy to modulate Fas-induced or tomodulate or regulate cellular functions such as apoptosis and immunedisorders mediated by the Fas pathway. The terms “Fas⁺ cellularfunction” is intended to mean cellular functions which are affected bythe binding of the receptor to its extracellular ligands, i.e., alone orin combination with each other. In some instances, it is desireable toaugment Fas⁺ function to induce apoptosis by introducing into the cellFADD protein or FADD nucleic acid. In other instances, it is desirableto down-regulate Fas⁺ cellular function by introducing into the cell aanti-FADD antibody or a nucleic acid encoding an anti-FADD antibody oralternatively, a FADD fragment or nucleic acid encoding it which is adominant negative inhibitor of functionally intact native FADD. Thistherapy will inhibit or disable intracellular Fas signaling andtherefore is a useful therapy where apoptotic cell death is to beavoided, such as in an HIV-infected T cell.

When used for gene therapy, a pharmaceutically acceptable vector ispreferred, such as a replication-incompetent retroviral vector. As usedherein, the term “pharmaceutically acceptable vector” includes, but isnot limited to, a vector or delivery vehicle having the ability toselectively target and introduce the nucleic acid into dividing cells.An example of such a vector is a “replication-incompetent” vectordefined by its inability to produce viral proteins, precluding spread ofthe vector in the infected host cell. An example of areplication-incompetent retroviral vector is LNL6 (Miller, A. D. et al.,(1989) BioTechniques 7:980-990). The methodology of usingreplication-incompetent retroviruses for retroviral-mediated genetransfer of gene markers is well established (Correll, et al. (1989),PNAS USA 86:8912; Bordignon, (1989), PNAS USA 86:8912-52; Culver, K.,(1991), PNAS USA 88:3155; and Rill, D. R. (1991), Blood 79(10):2694-700.Clinical investigations have shown that there are few or no adverseeffects associated with the viral vectors, see Anderson, (1992), Science256:808-13.

Antibodies

Also provided by this invention is an antibody capable of specificallyforming a complex with FADD protein or a fragment thereof, as well asnucleic acids encoding them. Vectors and host cells containing thesenucleic acids also are encompassed by this invention. The term“antibody” includes polyclonal antibodies and monoclonal antibodies. Theantibodies include, but are not limited to mouse, rat, rabbit or humanantibodies.

As used herein, an “antibody or polyclonal antibody” means a proteinthat is produced in response to immunization with an antigen orreceptor. The term “monoclonal antibody” means an immunoglobulin derivedfrom a single clone of cells. All monoclonal antibodies derived from theclone are chemically and structurally identical, and specific for asingle antigenic determinant. The hybridoma cell lines producing themonoclonal antibodies also are within the scope of this invention.

Laboratory methods for producing polyclonal antibodies and monoclonalantibodies, as well as deducing their corresponding nucleic acidsequences, are known in the art, see Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1988) andSambrook et al. (1989) supra. The monoclonal antibodies of thisinvention can be biologically produced by introducing FADD or a fragmentthereof into an animal, e.g., a mouse or a rabbit. The antibodyproducing cells in the animal are isolated and fused with myeloma cellsor heteromyeloma cells to produce hybrid cells or hybridomas.Accordingly, the hybridoma cells producing the monoclonal antibodies ofthis invention also are provided.

Thus, using the FADD protein or fragment thereof, and well knownmethods, one of skill in the art can produce and screen the hybridomacells and antibodies of this invention for antibodies having the abilityto bind FADD.

If a monoclonal antibody being tested binds with FADD protein, then theantibody being tested and the antibodies provided by the hybridomas ofthis invention are equivalent. It also is possible to determine withoutundue experimentation, whether an antibody has the same specificity asthe monoclonal antibody of this invention by determining whether theantibody being tested prevents a monoclonal antibody of this inventionfrom binding FADD with which the monoclonal antibody is normallyreactive. If the antibody being tested competes with the monoclonalantibody of the invention as shown by a decrease in binding by themonoclonal antibody of this invention, then it is likely that the twoantibodies bind to the same or a closely related epitope. Alternatively,one can pre-incubate the monoclonal antibody of this invention with FADDprotein with which it is normally reactive, and determine if themonoclonal antibody being tested is inhibited in its ability to bind theantigen. If the monoclonal antibody being tested is inhibited then, inall likelihood, it has the same, or a closely related, epitopicspecificity as the monoclonal antibody of this invention.

The term “antibody” also is intended to include antibodies of allisotypes. Particular isotypes of a monoclonal antibody can be preparedeither directly by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof different isotype by using the sib selection technique to isolateclass switch variants using the procedure described in Steplewski et al.(1985) Proc. Natl. Acad. Sci. 82:8653 or Spira et al. (1984) J. Immunol.Methods 74:307.

This invention also provides biological active fragments of thepolyclonal and monoclonal antibodies described above. These “antibodyfragments” retain some ability to selectively bind with its antigen orimmunogen. Such antibody fragments can include, but are not limited to:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule produced by digestion with the enzymepapain to yield an intact light chain and a portion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule obtained by treating withpepsin, followed by reduction, to yield an intact light chain and aportion of the heavy chain; two Fab′ fragments are obtained per antibodymolecule;

(3) (Fab′)₂, the fragment of the antibody that is obtained by treatingwith the enzyme pepsin without subsequent reduction; F(ab′)₂ is a dimerof two Fab′ fragments held together by two disulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) SCA, defined as a genetically engineered molecule containing thevariable region of the light chain, the variable region of the heavychain, linked by a suitable polypeptide linker as a genetically fusedsingle chain molecule.

A specific examples of “biologically active antibody fragment” includethe CDR regions of the antibodies. Methods of making these fragments areknown in the art, see for example, Harlow and Lane, (1988) supra.

The antibodies of this invention also can be modified to create chimericantibodies (Oi, et al. (1986) BioTechniques 4(3):214). Chimericantibodies are those in which the various domains of the antibodies'heavy and light chains are coded for by DNA from more than one species.

The isolation of other hybridomas secreting monoclonal antibodies withthe specificity of the monoclonal antibodies of the invention can alsobe accomplished by one of ordinary skill in the art by producinganti-idiotypic antibodies (Herlyn, et al., Science, 232:100, 1986). Ananti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the monoclonal antibody produced by thehybridoma of interest. These determinants are located in thehypervariable region of the antibody. It is this region which binds to agiven epitope and, thus, it is responsible for the specificity of theantibody. The anti-idiotypic antibody can be prepared by immunizing ananimal with the monoclonal antibody of interest. The animal immunizedwill recognize and respond to the idiotypic determinants of theimmunizing antibody by producing an antibody,to these idiotypicdeterminants. By using the anti-idiotypic antibodies of the secondanimal, which are specific for the monoclonal antibodies produced by asingle hybridoma which was used to immunize the second animal, it is nowpossible to identify other clones with the same idiotype as the antibodyof the hybridoma used for immunization.

Idiotypic identity between monoclonal antibodies of two hybridomasdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing antibodies to the epitopic determinants on a monoclonal antibodyit is possible to identify other hybridomas expressing monoclonalantibodies of the same epitopic specificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is themirror image of the epitope bound by the first monoclonal antibody.Thus, in this instance, the anti-idiotypic monoclonal antibody could beused for immunization for production of these antibodies.

As used in this invention, the term “epitope” is meant to include anydeterminant having specific affinity for the monoclonal antibodies ofthe invention. Epitopic determinants usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

Also encompassed by this invention are proteins or polypeptides thathave been recombinantly produced, biochemically synthesized, chemicallysynthesized or chemically modified, that retain the ability to bindFADD, the intracellular binding domain of the Fas receptor, or afragment thereof, as the corresponding native polyclonal or monoclonalantibody.

The antibodies of this invention can be linked to a detectable agent ora hapten. The complex is useful to detect the Fas receptor or FADDprotein or fragments in a sample or detect agents which interfere withFADD-Fas receptor binding, using standard immunochemical techniques suchas immunohistochemistry as described by Harlow and Lane (1988) supra.Examples of types of immunoassays which can utilize monoclonalantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the enzyme linked immunoassay (ELISA) radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of using themonoclonal antibodies of the invention can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation.

Another technique which may also result in greater sensitivity consistsof coupling the antibodies to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactsavidin, or dinitropherryl, pyridoxal, and fluorescein, which can reactwith specific anti-hapten antibodies. See Harlow and Lane (1988) supra.

The monoclonal antibodies of the invention can be bound to manydifferent carriers. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding monoclonal antibodies, or will beable to ascertain such, using routine experimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds, andbioluminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the monoclonal antibody, or willbe able to ascertain such, using routine experimentation. Furthermore,the binding of these labels to the monoclonal antibody of the inventioncan be done using standard techniques common to those of ordinary skillin the art.

For purposes of the invention, FADD may be detected by the monoclonalantibodies of the invention when present in biological fluids andtissues. Any sample of Fas³⁰ cell or tissue lysate containing adetectable amount of FADD can be used.

Compositions

This invention also provides compositions containing any of theabove-mentioned proteins, muteins, polypeptides or fragments thereof,and an acceptable solid or liquid carrier. When the compositions areused pharmaceutically, they are combined with a “pharmaceuticallyacceptable carrier” for administration. As used herein, the term“pharmaceutically acceptable carrier” encompasses any of the standardpharmaceutical carriers, such as a phosphate buffered saline solution,water, and emulsions, such as an oil/water or water/oil emulsion, andvarious types of wetting agents. The compositions also can includestabilizers and preservatives. For examples of carriers, stabilizers andadjuvants, see Martin, Remington's Pharm. Sci., 15th Ed. (Mack Publ.Co., Easton (1975)). These compositions can be used for the preparationof medicaments for the diagnosis and treatment of pathologies associatedwith the loss of the Fas receptor pathway.

Utilities

The use of the compositions and methods in vitro provides a powerfulbioassay for screening for drugs which are agonists or antagonists ofFas-FADD pathway in Fas⁺ cells. A Fas⁺ cell is one which contains theFas receptor or which is induced to PCD by an endogenous agent such asHIV, anti-TCR antibody, TNF and anti-Fas antibody. In one embodiment,these cells constitutively and inducibly express receptors for either orboth of the cytokine tumor necrosis factor (TNF) or the cell deathtransducing receptor Fas or TCR and which have been activated by theirrespective ligand. Recently, three separate groups have reported thatFas-induced apoptosis is involved in T cell death. Specifically, onegroup has shown that the Fas receptor, which can transduce a potentapoptotic signal when ligated, is rapidly expressed following activationon T cell hybridomas. It was suggested that the Fas receptor-ligandinteraction induces cell death in a cell-autonomous manner. See Dhein etal. (1995) Nature 373:438-441; Brunner et al. (1995)Nature 373:441-444;and Ju et al. (1995) Nature 373:444-448.

For the purpose of illustration only, examples of suitable cells are Tlymphocytes (T cells) (e.g., TCR⁺, CD4⁺ and CD8⁺ T cells) leukocytes andmixed leukocyte cultures (MLC), B lymphoma cells (e.g., A202J (ATCC)),bone marrow cells, endothelial cells, breast carcinoma cells, fibroblastcells, epithelial tumor cells (see Spriggs, D. R. et al. (1988) J. Clin.Inves. 81:455-460) and monocytes. Because Fas (APO-1/CD95) cell surfaceis a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF)receptor superfamily, any cell having a receptor of this family isintended to be encompassed by the scope of this invention. Fas and TNFreceptor expression also has been identified on numerous tissues, seefor example Watanabe-Fukunaga et al. (1992) J. Immun. 148:1049-1054 andOwen-Schaub, L. B. et al. (1994) Cancer Res. 54:1580-1586; Dhein et al.(1995) Nature 373:438-441; Brunner et al. (1995) Nature 373:441-444; andJu et al. (1995) Nature 373:444-448. Assays for identifying additional“suitable” cells sensitive to induction or activation, e.g., TCR-, TNF-or Fas-related apoptosis, are well known to those of skill in the art.(See for example, Opipairi, et al. J. Biol. Chem. (1992)267:12424-12427; Yonehara et al. J. Exp. Med. (1989) 169:1747-1756;Dhein et al. (1995) supra; Brunner et al. (1995) supra and Ju et al.(1995) supra) However, this method is particularly suitable for use withTCR⁺, CD8⁺ or CD4⁺ T cells or tissues that harbor the simianimmunodeficiency virus (SIV) or alternatively, the humanimmunodeficiency virus (HIV). The cells can be mammalian cells or animalcells, such as guinea pig cells, rabbit cells, simian cells, mousecells, rat cells, or human cells. They can be continuously cultured orisolated from an animal or human. In a separate embodiment of thisinvention, neurological cells are specifically excluded.

It also provides a powerful assay to determine whether an agent ofinterest, such as a pharmaceutical, is useful to treat a Fas-mediateddisorder or to further augment or disable Fas receptor function. Forexample, the composition to be tested can be added prior to,simultaneously or subsequent to FADD as described above. A separate“control” assay is run simultaneously under the same conditions butwithout the addition of the composition or drug being tested. If theagent inhibits binding of FADD to the intracellular Fas domain (ascompared to control) the agent is a candidate for inhibiting orpreventing Fas receptor mediated function in the cell, tissue orsubject.

More specifically, the in vitro method comprises providing cell culturesor tissue cultures having either a cell surface receptor that mediatesapoptosis such as a TCR, the TNF receptor or the Fas receptor. The cellsare cultured under conditions (temperature, growth or culture medium andgas (CO₂)) and for an appropriate amount of time to attain exponentialproliferation without density dependent constraints. The cells are thenexposed to preliminary conditions necessary for apoptosis, for examplean effective amount of an inducing agent, e.g., a TCR ligand, HIV, SIV,TNF, or a Fas ligand such as an anti-Fas antibody is added to theculture. Anti-Fas antibodies and mitogens (ConA) are well known to thoseof skill in the art. (Itoh, N. et al. (1991) Cell 66:233-243 andYonehara et al. (1989) J. Exp. Med. (1989) 169:1747-1756). These cellsare now “induced” to apoptosis. The cells are again cultured undersuitable temperature and time conditions. In one embodiment, HIV or SIVis added to the culture. In other embodiments, a drug or agent to betested is added in varying concentrations at a time that is simultaneouswith, prior to, or after the inducing agent.

The FADD nucleic acid or protein is then added to the culture in aneffective amount and the cells are cultured under suitable temperatureand time conditions to induce apoptosis. The FADD nucleic acid orprotein can be added prior to, simultaneously with, or after, theinducing agent. The cells are assayed for apoptotic activity usingmethods well known to those of skill in the art and described herein. Itis apparent to those of skill in the art that two separate culture ofcells must be treated and maintained as the test population. One ismaintained without receiving an inducing agent to determine backgroundrelease and the second without receiving the agent to be tested. Thesecond population of cells acts as a control.

The use of the compositions and methods in vitro provides a powerfulbioassay for screening for drugs which are agonists or antagonists ofFas mediated cellular function in these cells. Thus, one can screen fordrugs having similar or enhanced ability to prevent or inhibitapoptosis. The in vitro method further provides an assay to determine ifthe method of this invention is useful to treat a subject's pathologicalcondition or disease that has been linked to apoptotic cell death in theindividual.

When the method is practiced in vivo in a human patient, it isunnecessary to provide the inducing agent since it is provided by thepatient's immune system. However, when practiced in an experimentalanimal model, it can be necessary to provide an effective amount of theinducing agent in a pharmaceutically acceptable carrier prior toadministration of the FADD or anti-FADD product, to induce apoptosis.When the method is practiced in vivo, the carrying vector, polypeptide,polypeptide equivalent, or expression vector can be added to apharmaceutically acceptable carrier and systemically administered to thesubject, such as a human patient or an animal such as a mouse, a guineapig, a simian, a rabbit or a rat. Alternatively, it can be directlyinfused into the cell by microinjection. A fusion protein also can beconstructed comprising the T-cell specific ligand for targeting to a Tcell. Such T cell specific ligands include, but are not limited toanti-CD3, anti-CD4, anti-CD28 and anti-IL-1 antibody protein.

Accordingly, this invention also provides a method for screening for anagent to regulate the Fas receptor pathway, comprising the steps of: a)providing a Fas cytoplasmic domain receptor bound to a solid support; b)contacting the agent to be tested with the receptor bound support ofstep a) under conditions favoring binding of the cytoplasmic domain tothe receptor to FADD; c) contacting detectably-labeled FADD to the solidsupport of step b) under conditions favoring binding of Fas cytoplasmicdomain receptor to FADD; d) detecting the presence of any complex formedbetween the Fas receptor and FADD to form Fas receptor-FADD complex; ande) the absence of complex being indicative that the agent inhibitsbinding of FADD to the Fas receptor and therefore is an agent to inhibitFas mediated function such as apoptosis.

This invention provides an alternative method for screening for a FADDor Fas-receptor immunosuppressive agent, which comprises the steps of a)providing a Fas cytoplasmic domain receptor bound to a solid support;b)contacting detectably-labeled FADD to the solid support of step a) underconditions favoring binding of the cytoplasmic domain receptor to FADD;c) contacting the agent to be screened with the receptor bound supportof step b) under conditions favoring binding of the cytoplasmic domainto the receptor to FADD; d) detecting the presence of any complex formedbetween Fas receptor and FADD to form Fas receptor-FADD complex; and e)the absence of complex being indicative that the agent inhibits bindingof FADD to the Fas receptor and therefore is an inhibitor of Fasreceptor mediated cellular function. This invention also provides theagents detected by these methods and the use of these agents in thetherapeutic methods described herein. As is apparent to those of skillin the art, the above compositions can be combined with instructions foruse to provide a kit for a commercially available screen.

The compositions provided herein also are useful to modulate the Fasreceptor pathway and cellular functions associated with this pathway,for example, preventing or inhibiting Fas regulated apoptosis or growthand differentiation of cells. As used herein, the term “Fas-receptormediated or modulated cellular function” is to include any cellularresponse or function which has been linked to the binding of Fas orFas/TNF receptor complex to its extracellular and/or intracellularligand.

When applied to apoptosis, the terms “preventing” or “inhibiting” areintended to mean a reduction in cell death or a prolongation in thesurvival time of the cell. They also are intended to mean a diminutionin the appearance or a delay in the appearance of morphological and/orbiochemical changes normally associated with apoptosis. Thus, thisinvention provides compositions and methods to increase survival timeand/or survival rate of a cell or population of cells which, absent theuse of the method, would normally be expected to die. Accordingly, italso provides compositions and methods to prevent or treat diseases orpathological conditions associated with unwanted cell death in asubject.

Apoptosis can be assessed by the use of fluorescent DNA-staining dyes toreveal nuclear morphology and by transmission electron microscopy. Forpropidium iodide staining, cells can be grown on 22 mm² No. 1 glasscoverslips (Corning) placed in 35 mm wells of a 6-well culture dish(Costar). Following treatment with TNF, anti-Fas cycloheximide (CHX), orno treatment, medium can be removed and the wells rinsed twice withphosphate buffered saline (PBS), fixed in 100% methanol at −20° C. for10 minutes, washed three times with PBS, and stained at room temperaturefor 10 minutes in a 100 μg/ml solution or propidium iodide (Sigma) madein PBS. The coverslips are then washed three times with PBS, blotted dryand mounted onto glass slides using Vectashield mounting medium forfluorescence (Vector Laboratories). Cells can be stained using acridineorange (sigma) by preparing a wet mount of 30 μl of a cell suspension ata density of approximately 3×10⁵ cell/ml mixed with 5 μl of a 100 μg/mlacridine orange solution made in PBS. Both propidium iodide-stained MCF7and acridine orange-stained BJAB nuclei were visualized by fluorescencemicroscopy using a FITC range barrier filter cube. Laser-scanningconfocal microscopy was performed using the Bio-Rad MRC 600 confocalmicroscope and digitized images obtained were artificially colorized.

For electron microscopy, cells can be fixed and processed as perstandard electron microscopy procedures.

When a function associated with the Fas receptor mediated pathway shouldbe augmented, nucleic acid molecules coding for FADD can be insertedinto a Fas⁺ cell, such as a T cell, using an appropriate pharmaceuticalvector. Alternatively, when a function, associated with the Fas receptorpathway should be inhibited or prevented, a nucleic acid coding foranti-FADD antibody fragment, a dominant inhibitory FADD polypeptidefragment, or anti-sense FADD RNA can be introduced into a Fas⁺ cellusing an appropriate pharmaceutical vector.

When practiced in vivo, the compositions and methods are particularlyuseful for modulating or regulating Fas receptor induced function in asubject or an individual suffering from or predisposed to suffer fromreceptor-related disfunction, CD4 T cell depletion associated with HIVinfection. When the animal is an experimental animal such as a mouse,this method provides a powerful assay to screen for new drugs that maybe used alone or in combination with this invention to ameliorate orreduce the symptoms and infections associated withFas-related-disfunction such as CD4 T cell depletion.

As used herein, the term “administering” for in vivo purposes meansproviding the subject with an effective amount of the nucleic acidmolecule, polypeptide or antibody, effective to modulate Fas-relatedfunction of the target cell. Methods of administering pharmaceuticalcompositions are well known to those of skill in the art and include,but are not limited to, microinjection, intravenous or parenteraladministration. The compositions are intended for topical, oral, orlocal administration as well as intravenously, subcutaneously, orintramuscularly. Administration can be effected continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to those of skill in the art and will vary with the vectorused for therapy, the polypeptide or protein used for therapy, thepurpose of the therapy, the target cell being treated, and the subjectbeing treated. Single or multiple administrations can be carried outwith the dose level and pattern being selected by the treatingphysician.

The compositions also can be administered to subjects or individualssusceptible to or at risk of developing a Fas receptor induced disease.In one embodiment, the composition can be administered to a subjectsusceptible to the disease or pathology to maintain lymphocyte cellfunction such as antibody production. In these instances, a“prophylactically effective amount” of the composition is administeredwhich is defined herein to be an amount that is effective to maintainthe targeted cellular function, such as lymphocyte function, at anacceptable level.

It should be understood that by preventing or inhibiting Fas receptorrelated disfunction in a subject or individual, the compositions andmethods of this invention also provide methods for treating, preventingor ameliorating the symptoms associated with a Fas receptor mediateddisease.

When practiced in vivo, the compositions and methods are particularlyuseful for maintaining T cell viability and function in a subject or anindividual suffering from or predisposed to suffer from abnormallymphocyte death. When the animal is an experimental animal such as asimian (using SIV), this method provides a powerful assay to screen fornew drugs that may be used alone or in combination with this inventionto ameliorate or reduce the symptoms and opportunistic infectionsassociated with HIV infection or AIDS.

This invention also is particularly useful to ward off lymphocyte deathor immunosuppression in AIDS patients. By preventing or inhibitingapoptosis, not only is cell death prevented but functionality, e.g.,immuno-proliferative capacity, is restored to the cell and a responsiveimmune system is retained or regained. Accordingly, the compositions andmethods of this invention are suitably combined with compositions andmethods which prevent or inhibit HIV infectivity and replication.

The method can also be practiced ex vivo using a modification of themethod described in Lum et al. (1993) Bone Marrow Transplantation12:565-571. Generally, a sample of cells such as bone marrow cells orMLC can be removed from a subject or animal using methods well known tothose of skill in the art. An effective amount of FADD or anti-FADDnucleic acid is added to the cells and the cells are cultured underconditions that favor internalization of the nucleic acid by the cells.The transformed cells are then returned or reintroduced to the samesubject or animal (autologous) or one of the same species (allogeneic)in an effective amount and in combination with appropriatepharmaceutical compositions and carriers.

As used herein, the term “administering” for in vivo and ex vivopurposes means providing the subject with an effective amount of thenucleic acid molecule or polypeptide effective to prevent or inhibitapoptosis of the target cell. Methods of administering pharmaceuticalcompositions are well known to those of skill in the art and include,but are not limited to, microinjection, intravenous or parenteraladministration. The compositions are intended for topical, oral, orlocal administration as well as intravenously, subcutaneously, orintramuscularly. Administration can be effected continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration arewell known to those of skill in the art and will vary with the vectorused for therapy, the polypeptide or protein used for therapy, thepurpose of the therapy, the target cell being treated, and the subjectbeing treated. Single or multiple administrations can be carried outwith the dose level and pattern being selected by the treatingphysician. For example, the compositions can be administered prior to asubject already suffering from a disease or condition that is linked toapoptosis. In this situation, an effective “therapeutic amount” of thecomposition is administered to prevent or at least partially arrestapoptosis and the accompanying pathology such as immunosuppression inHIV infected individuals.

However, the compositions can be administered to subjects or individualssusceptible to or at risk of developing apoptosis-related disease toprevent pathological cell death. In one embodiment, the composition canbe administered to a subject susceptible to HIV-related lymphocytedisfunction to maintain lymphocyte cell function and viability. In thesea “prophylactically effective amount” of the composition is administeredto maintain cellular viability and function at a level near to thepre-infection level.

It should be understood that by preventing or inhibiting unwanted celldeath in a subject or individual, the compositions and methods of thisinvention also provide methods for treating, preventing or amelioratingthe symptoms associated with a disease characterized by apoptosis ofcells. Such diseases include but are not limited to AIDS, acute andchronic inflammatory disease, leukemia, myocardial infarction, stroke,traumatic brain injury, neural and muscular degenerative diseases,aging, tumor induced-cachexia and hair loss.

This invention also provides vector and protein compositions useful forthe preparation of medicaments which can be used for preventing orinhibiting apoptosis, maintaining cellular function and viability in asuitable cell or for the treatment of a disease characterized by theunwanted death of target cells.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand the following examples are intended to illustrate and not limit thescope of the invention. Other aspects, advantages and modificationswithin the scope of the invention will be apparent to those skilled inthe art to which the invention pertains.

EXPERIMENTAL PROCEDURES Experiment I

The following yeast two-hybrid system was used and constructed asfollows. The cytoplasmic domains of Fas, Fas-FD8, TNRF-1, Δ-TNFR-1,CD40, and CD28 were obtained by PCR and cloned in-frame, as confirmed bysequencing, into the GAL4 DNA binding domain (GAL4bd) vector pAS1CYH2.Full-length A20 and B94 were similarly cloned into the bait vector.GAL4bd-Fas was cotransformed with a prey plasmid containing a humanB-cell cDNA expression library fused to the GAL4 activation domain(GAL4ad) in the pACT plasmid. A more detailed account of the plasmidsused in the procedure for the yeast two-hybrid system can be found in Huet al. (1994) J. Biol. Chem. 269:30069-30072.

The yeast two-hybrid system was used to screen for proteins thatinteract with the cytoplasmic domain of Fas. An expression vector wasconstructed by fusing the GAL4 DNA-binding domain to the cytoplasmictail of the human Fas antigen (GAL4bd-Fas). This bait plasmid wascotransformed in yeast with a prey plasmid containing a human B-cellcDNA expression library fused to the GAL4-activation domain. Seventeenpositive clones were obtained from 2×10⁶ transformants screened. Todetermine the specificity of interaction, plasmids containing theactivation domain fusion proteins were recovered from the putativepositive clones and cotransformed with GAL4bd-Fas and controlheterologous baits. Two clones (8 and 15) were found to interact withthe GAL4. DNA-binding domain fusion protein containing the cytoplasmicdomain of wild-type Fas and not the functionally inactive deletionmutant, Fas-FD8 (Itoh et al., 1993) or the indicated heterologous baits(FIG. 1).

Experiment II

Isolation of the Sequence

Double-stranded plasmid template was sequenced on both strands by thedideoxy chain termination method using modified T7 DNA polymerase(Sequenase, U.S. Biochemical Corp.). Manual sequencing was confirmed bysubsequent automated sequencing. Network BLAST searches were conductedusing the NCBI-online service. Sequences were compared using theMogAlign (DNASTAR) software.

A random-promed cDNA library was constructed in the pcDNA1 vector(Invitrogen) from TNF/cycloheximide treated human umbilical veinendothelial cell poly(A)⁺ RNA. 5×10⁵ colonies were screened with a ³²Prandom-labeled XhoI restriction fragment of the yeast prey plasmidencoding GAL4ad-FADD (clone 15) using standard techniques (Sambrook etal. (1989) supra.

As noted in Experiment I, clones 8 and 15 isolated by the yeasttwo-hybrid screen were found to contain overlapping sequence fused tothe GAL4 activation domain in the same reading frame. To obtain afull-length coding sequence, a human umbilical vein endothelial cell(HUVEC) library was screened with a cDNA insert obtained from clone 15.Two independent clones yielded a 1.6 kb cDNA containing an open readingframe that begins with an initiator methionine conforming to Kozak'sconsensus (Kozak, 1989) and that ends 625 nucleotides later at an Opalcodon. Given the presence of an in-frame stop codon 130 base pairsupstream of the initiator methionine and the size of the transcript(˜1.6 kb; FIG. 3), it is likely that FIG. 2A represents the full-lengthcoding sequence. This gene encodes a novel protein of 208 amino acidswith a predicted molecular weight of 23.3 kDa, designated FADD.

A BLAST search revealed that residues 111-170 of FADD matched residues233-292 of rat Fas antigen (rFas, p=0.0012) and shared 27% identity (51%of the amino acids were conserved). This region in the cytoplasmicdomain of rFas corresponds to the death domain, a region of homologyshared by both Fas and TNFR-1 that signals cell death (Tartaglia et al.,1993; Itoh et al., 1993).

Dependent upon the alignment and boundaries selected, the death domainsof FADD, Fas, and TNFR-1 share 25-30% identity (FIG. 2B). Whenconservative amino acid substitutions are included, the homologiesapproach 50%. These numbers are consistent with those previouslyreported for the death domain homology between TNFR-1 and Fas (Tartagliaet al. (1993) supra Itoh et al. (1993) supra). Interestingly, V¹²¹ ofFADD is aligned and conserved with V²³⁸ of Fas, which when altered to anasparagine, abolishes the cell killing activity of Fas and in mice, isresponsible for the lymphoproliferation (Lpr) phenotype(Watanabe-Fukunaga et al., 1992). A corresponding inactivating mutationalso exists in TNFR-1, L³⁵¹→N³⁵¹ (Tartaglia et al., 1993)

Experiment III

Northern Blot Analysis of Tissues

Adult and fetal human multiple tissue Northern blots (CLONTECH) werehybridized, according to the manufacturer's instructions usingradiolabeled cDNA insert obtained from an XhoI digestion of the yeastprey plasmid encoding GAL4ad-FADD (clone 15).

Northern blot analysis revealed that FADD is constitutively expressed ina wide array of fetal and adult human tissues (FIG. 3). The mRNAtranscript is approximately 1.6 kb, consistent with the size of the cDNAclones isolated from the HUVEC library.

Experiment IV

FADD was cloned into pcDNA3 (Invitrogen) in which an HA-epitope tag(YPYDVPDYA) (SEQ ID No:7) had previously been placed downstream of thecytomegalovirus promoter/enhancer (pcDNA3 HA-FADD). In addition, anAU1-epitope (DTYRYI) (SEQ ID NO:8) tagged FADD was made with PCR primersencoding the epitope and using the FADD cDNA as template (pcDNA3AU1-FADD). FLAG (DYKDDDDK) (SEQ ID NO:9)-tagged constructs of Fas andmutants were also made in pcDNA3 using full-length Fas as a template.The 5° FLAG PCR primer was engineered to encode a FLAG epitope 5 aminoacids downstream of the putative signal sequence site of Fas and is asfollows: AAG CCT GGT ACC ATG CTG GGC ATC TGG ACC CTC CTA CCT CTG GTT CTTACG TCT GTT GCT AGA TTA TCG TCC AAA GAC TAC AAG GAC GAC GAT GAC AAG AGTGTT AAT GCC CAA GTC (SEQ ID NO:10). The amplified products were thencloned into the KpnI/XhoI site of pcDNA3. pcDNA3 AU1-FADDmt and pcDNA3FLAG-Fas-LPR were made by site-directed mutagenesis using a two-step PCRprotocol as described in Higuchi, R. et al. (1988) Nucleic Acids Res.16:7351-7367. The V¹²¹→N¹²¹ and V²³⁸→N²³⁸ mutations, respectively, wereconfirmed by sequence analysis.

Experiment V

GST Fusion Protein Expression and In Vitro Binding Assay

The cytoplasmic domains of Fas, Fas-FD5, Fas-FD8, and TNFR-1 wereamplified by PCR using appropriate templates and primers and clonedin-frame into pGSTag using the method disclosed in Ron, D. et al. (1992)Biotechnigues 13:866-869. Fas-LPft was made by site-directed mutagenesisusing a two-step PCR protocol (Higuchi et al., 1988) and cloned intopGSTag. The V²³⁸→N²³⁸ mutation was confirmed by sequence analysis. ThepGSTag constructs were then transformed into the E. coli strainBL21(DE3)pLysS (Studier, 1991). GST and GST fusions were prepared usingpublished procedures (Studier, (1991) J. Mol. Biol. 219:37-44) and therecombinant proteins immobilized onto glutathione-agarose beads asdescribed Harper, J. W. et al. (1993) Cell 75:805-816.

Labeled FADD was prepared by in vitro transcription/translation usingTNT T7 coupled reticulocyte lysate system from Promega according to themanufacturer's instructions, using pcDNA3 HA-FADD as template.

Following translation, equal amounts of total ³⁵S-labeled reticulocytelysate were diluted into 150 μl GST binding buffer (50 mM Tris, pH 7.6,120 mM NaCl, 1% Brij) and incubated for 2 hrs. at 4° C. with the variousGST fusion proteins complexed to beads, following which the beads werepelleted by pulse centrifugation, washed 3 times in GST buffer, boiledin SDS-sample buffer and resolved on a 10% SDS-acrylamide gel. Boundproteins were visualized following autoradiography at −80° C.

Lysates of FADD or FADDmt-transfected 293T cells were processed as aboveexcept that the GST binding buffer also had 10% glycerol and a proteaseinhibitor cocktail. For some experiments, the complexed GST beads weredissociated by boiling in PBS+1% SDS, diluted tenfold in PBS containing1% deoxycholate and subsequently subjected to immunoprecipitationanalysis.

Experiment VI

Transfection, metabolic labeling and immunoprecipitation analysis wasperformed as described in O'Rourke, K. M. et al. (1992) J. Biol. Chem.267:24921-24924. For re-immunoprecipitation analysis, the initial immunecomplex was dissociated by boiling in PBS+1% SDS, diluted tenfold in PBScontaining 1% deoxycholate and subjected to a second round ofimmunoprecipitation analysis.

To confirm the interaction observed in yeast, radiolabeled in vitrotranslated FADD was precipitated with various GST fusion proteinsimmobilized on glutathione-Sepharose beads (FIGS. 4A, B). As predicted,FADD specifically associated with GST-Fas but, not GST, GST-Fas-FD8, orGST-Fas-LPR, which contains the cytoplasmic domain of the functionallyinactive point mutant of Fas (Itoh et al., 1993). A very weakinteraction was observed between FADD and TNFR-1. Interestingly,relative to its association with GST-Fas, FADD strongly interacted withGST-Fas-FD5, which is a 15 amino acid C-terminal deletion mutant of Faspossessing enhanced killing activity (Itoh et al., 1993). Similarresults were obtained when detergent lysates of 293T cells expressingFADD were precipitated with the various GST fusion proteins (FIG. 4C).

Experiment VII

Functional Assay and Immunocytochemistry

Stable CrmA and vector transfectants (BJAB) were described previously(Tewari et al. (1995) supra.). For transient transfections, 5×10⁶ cellswere electroporated at 220V, 960 μF in 0.4 cm cuvettes (Bio-Rad) using20 μg of pCMV β-galactosidase ±30 μg ofpcDNA3 AU-FADD. After 12 hours,cells were cytocentrifuged, fixed with 1% paraformaldehyde,permeabilized with 0.1% Triton/PBS, blocked with horse serum, andincubated with rabbit anti-β-galactosidase (1:200 dilution, Cappel) for1 hour. The cells were subsequently washed with PBS, incubated withbiotinylated anti-rabbit antibody (1:200 dilution, Vector Laboratories)for 20 min., washed with PBS, and incubated with Avidin-FITC (1:100dilution, Vector Laboratories) for 20 min. The nuclei were stained witha 10 μg/ml solution of propidium iodide (Sigma) for 10 minutes. Cellswere visualized by fluorescence microscopy using aFITC range barrierfilter cube. For graphical data, at least 100 β-galactosidase positivecells were counted for each transfection (n=3) and designated asapoptotic or non-apoptotic. Immunostaining for AU1-FADD was done asabove except that cells were fixed in 100% methanol at −20ΣC for 10min., the primary antibody was against the AU1 epitope (1:50 dilution,Babco) and the secondary antibody was a FITC conjugated anti-mouse Ab(Sigma).

Coimmunoprecipitation of FADD and Fas

To demonstrate the interaction of FADD and Fas in vivo, 293T cells weretransiently transfected with HA-epitope tagged FADD (HA-FADD) andFLAG-epitope tagged Fas (FLAG-Fas) and mutants (FIG. 5). Expression ofthe FLAG-tagged constructs was shown by immunoprecipitation with ananti-FLAG (α-FLAG) antibody (FIG. 5B). Likewise, immunoprecipitationwith anti-HA (α-HA) antibody showed expression of HA-FADD, and asexpected, FLAG-Fas and FLAG-Fas-FD5 individually coprecipitated, whilethe functionally inactive mutants, FLAG-Fas-FD8 and FLAG-Fas-LPR did not(FIG. 5C). The α-HA immunoprecipitates were dissociated and subjected toa second round of immunoprecipitation with α-FLAG antibody. Consistentwith results of the primary immunoprecipitation (with α-HA), a doubleimmunoprecipitation with α-HA followed by α-FLAG, confirmed the presenceof FLAG-Fas and FLAG-Fas-FD5 in the original immune complexes (FIG. 5D).

The Death Domain of FADD Interacts With the Death Domain of Fas

Previous studies have reported that the death domains of TNFR-1 and Fasself-associate (Boldin et al., (1995) supra and Wallach et al. (1994)supra). The two clones (8 and 15) isolated in the two-hybrid screendescribed above (using the cytoplasmic domain of Fas as bait) did notcontain various portions of the N-terminus of wild-type FADD. Theshortest of the two, clone 8, is missing the N-terminal 40 amino acids,suggesting that the C-terminal half of FADD, which contains the deathdomain, is interacting with the cytoplasmic tail of Fas. Morespecifically, our results show that FADD interacts with death domain ofFas, since it fails to associate with Fas-LPR and Fas-FD8, a pointmutant and deletion mutant, respectively, of the Fas death domain.

Thus, it is reasonable to propose that the death domain of FADD isinteracting with its homologous counterpart in Fas. To test thishypothesis, a point mutant of FADD (FADDmt) was engineered in which V¹²¹is altered to an asparagine. This mutation corresponds to theinactivating Lpr mutation (V²³⁸→N²³⁸) of Fas and the L³⁵¹→N³⁵¹ mutationof TNFR-1. 293T cells were transiently transfected with expressionconstructs containing AU1-epitope tagged FADD (AU1-FADD) and AU1-FADDmt.Detergent lysates were prepared and subsequently precipitated with GST,GST-Fas and GST-Fas-LPR immobilized on glutathione-Sepharose beads (FIG.6). As predicted, AU1-FADD bound GST-Fas and not GST or GST-Fas-LPR,while in contrast, AU1-FADDmt failed to bind any of the GST fusions.Taken together, these results show that a death domain to death domaininteraction is responsible for the association of FADD and Fas.

Overexpression of FADD Initiates Apoptosis Which is Suppressed by CrmA.

To study the functional role of FADD the B-cell lymphoma cell line, BJABwas chosen. This is an ideal cell system to study proteins involved inFas signal transduction because BJAB cells are exquisitely sensitive toanti-Fas antibody induced cell death in the absence of protein synthesisinhibitors (Tewari et al. (1995) supra). Two well characterized clonalcell lines of BJAB were used in this study: one expresses CrmA, whichhas been shown to potently block Fas-mediated cell death, while theother is a corresponding vector control cell line (Tewari et al. (1995)supra). To help identify transiently transfected cells, the plasmid wasco-transfected with an expression construct encoding: β-galactosidase(pCMV β-gal). As expected, over 90′ of the cells that expressedβ-galactosidase also expressed the protein of interest as confirmed byimmunostaining.

CrmA-expressing and vector control BJAB cell lines were transfected withthe pCMV β-gal reporter in the presence or absence of an equimolaramount of an expression construct encoding AU1-epitope tagged FADD(pcDNA3 AU1-FADD). As expected, expression of β-galactosidase alone inboth the CrmA and vector clones did not induce apoptotic cell death asassessed by propidium iodide staining of nuclei of β-galactosidasepositive cells (FIG. 7A, upper panels). In contrast, however, the vectorcontrol cell line co-transfected with PCMV β-gal and pcDNA3 AU1-FADDexhibited prominent apoptotic morphology including chromatincondensation and cellular shrinkage (FIG. 7A, lower left panel). Moreimportantly, FADD-induced apoptosis, like Fas-induced apoptosis, wasinhibited in the CrmA-expressing line (FIG. 7A, lower right panel). Agraphical representation of this data is shown in. FIG. 7B. In thevector control lines, over 90% of the transfected cells expressing FADDwere apoptotic while less than 10% exhibited similar morphology in thecorresponding CrmA-expressing lines. As a control, expression ofAU1-TRAF1 and HA-CD40bp revealed less than 10% apoptotic morphology ineither the CrmA or vector cell lines. Immunostaining for AU1-FADD withan anti-AU1 antibody is shown in FIG. 7C. At present, it is unclearwhether FADD is a soluble cytoplasmic protein or associated withcellular membranes.

Experimental Summary

Using the yeast two-hybrid screen, FADD was identified as,a novelprotein that associates specifically with the cytoplasmic domain of Fas(FIG. 1). A BLAST search using the amino acid sequence of FADD revealeda stretch of 80 amino acids that were significantly homologous to thedeath domain of Fas (FIG. 2B). When the region of FADD was masked, theremaining sequences did not match any proteins in the database.Interestingly, BLAST searches using the death domains of FADD, Fas andTNFR-1 revealed a significant homology to the family of ankyrins(p<0.001 for all three death domains). More specifically, the respectivedeath domains aligned with approximately 80 amino acids of the negativeregulatory domain of ankyrin. A previous study reported that this regionof ankyrin is homologous to the cytoplasmic domain of TNFR-1 (Peters etal. (1993) Semin. in Hematol. 30:85-118), corroborating thisobservation. Why ankyrin contains a “death domain” remains unclear, butpresumably this region is acting as a protein interaction domain.

In vitro and in vivo studies show that FADD specifically associates withthe death domain of Fas, confirming the results of the yeast interactionassay. FADD failed to interact with Fas-LPR and Fas-FD8, a non-signalingpoint mutant and deletion mutant, respectively, of the Fas death domain.Interestingly, upon deletion of the negative regulatory domain of Fas,an enhanced interaction with FADD was observed. Hence, a correlationexists between the cell-killing activity of the various Fas mutants andtheir association with FADD (FIG. 5A). A weak association between FADDand TNFR-1 was observed in vitro (FIG. 4). In addition, β-galactosidasefilter assays of yeast cotransformed with GAL4bd-Fas and GAL4ad-FADDturned blue within 1 hr, while those cotransformed with GAL4bd-TNFR-1and GAL4ad-FADD turned blue overnight (the other cotransformedheterologous baits remained unchanged). If the weak interaction betweenFADD and TNFR-1 observed in yeast and in vitro proves to be significant,this would correlate with the relative potencies of Fas-dependent celldeath and TNF-dependent cytotoxicity (Clement, M-V. et al. (1994) J.Exp. Med. 180:557-567).

Having shown that FADD specifically binds the death domain of Fas, thenext step was to identify the corresponding interaction-domain in Fas.Previous studies have shown that death domains have a propensity toself-associate (Boldin et al. (1995) supra). It was thus reasonable topropose that the death domain of FADD was interacting with itshomologous counterpart in Fas. As predicted, a point mutation in thedeath domain of FADD abrogated its association with Fas (FIG. 6). Theseresults support a model in which a death domain to death domaininteraction is responsible for the binding of FADD to Fas (FIG. 8).

Once the in vitro and in vivo association of FADD and Fas wasestablished, the next step was to determine a functional role for thisnovel Fas binding protein. BJAB cells transiently transfected withAU1-FADD undergo apoptosis within 12 hours—a time frame similar toFas-induced killing (FIGS. 7A and 7B). Previous studies showed that CrmAis a potent inhibitor of Fas-induced cell death (Tewari et al. (1995)supra). Likewise, CrmA suppressed FADD-induced cell death (FIGS. 7A and7B). These functional studies, together with the biochemical data,suggests that FADD is likely a component of the Fas-signal transductionmachinery.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand the examples are intended to illustrate and not limit the scope ofthe invention. Other aspects, advantages and modifications within thescope of the invention will be apparent to those skilled in the art towhich the invention pertains.

Throughout this application, various publications, patents and publishedpatent applications are referred to by an identifying citation. Thedisclosures of these publications, patents and published patentapplications are hereby incorporated by reference into this applicationto more fully describe the state of the art to which this inventionpertains.

10 1642 base pairs nucleic acid single linear cDNA CDS 130..753misc_feature 4..6 /note= “An in-frame stop codon 130 base pairs upstreamof the initiator methionine” polyA_signal 1636..1641 /note= “Potentialpoly(A) adenylation signal” misc_feature 198..753 /note= “Clone-15; 5′end of FADD” misc_feature 249..753 /note= “Clone-8; 5′ end of FADD”misc_feature 460..660 /note= “Death Domain of FADD” mutationreplace(490..492, “aay”) /note= “For FADDmt sequence is altered toeither AAT or misc_feature group(250..753, 232..753) /note= ”Codons cancomprise C-terminal polypeptide fragme misc_feature 460..639 /note=“death domain homology region” 1 CTCTAAAGGT TCGGGGGTGG AATCCTTGGGCCGCTGGGCA AGCGGCGAGA CCTGGCCAGG 60 GCCAGCGAGC CGAGGACAGA GGGCGCACGGAGGGCCGGGC CGCAGCCCCG GCCGCTTGCA 120 GACCCCGCC ATG GAC CCG TTC CTG GTGCTG CTG CAC TCG GTG TCG TCC 168 Met Asp Pro Phe Leu Val Leu Leu His SerVal Ser Ser 1 5 10 AGC CTG TCG AGC AGC GAG CTG ACC GAG CTC AAG TTC CTATGC CTC GGG 216 Ser Leu Ser Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu CysLeu Gly 15 20 25 CGC GTG GGC AAG CGC AAG CTG GAG CGC GTG CAG AGC GGC CTAGAC CTC 264 Arg Val Gly Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu AspLeu 30 35 40 45 TTC TCC ATG CTG CTG GAG CAG AAC GAC CTG GAG CCC GGG CACACC GAG 312 Phe Ser Met Leu Leu Glu Gln Asn Asp Leu Glu Pro Gly His ThrGlu 50 55 60 CTC CTG CGC GAG CTG CTC GCC TCC CTG CGG CGC CAC GAC CTG CTGCGG 360 Leu Leu Arg Glu Leu Leu Ala Ser Leu Arg Arg His Asp Leu Leu Arg65 70 75 CGC GTC GAC GAC TTC GAG GCG GGG GCG GCG GCC GGG GCC GCG CCT GGG408 Arg Val Asp Asp Phe Glu Ala Gly Ala Ala Ala Gly Ala Ala Pro Gly 8085 90 GAA GAA GAC CTG TGT GCA GCA TTT AAC GTC ATA TGT GAT AAT GTG GGG456 Glu Glu Asp Leu Cys Ala Ala Phe Asn Val Ile Cys Asp Asn Val Gly 95100 105 AAA GAT TGG AGA AGG CTG GCT CGT CAG CTC AAA GTC TCA GAC ACC AAG504 Lys Asp Trp Arg Arg Leu Ala Arg Gln Leu Lys Val Ser Asp Thr Lys 110115 120 125 ATC GAC AGC ATC GAG GAC AGA TAC CCC CGC AAC CTG ACA GAG CGTGTG 552 Ile Asp Ser Ile Glu Asp Arg Tyr Pro Arg Asn Leu Thr Glu Arg Val130 135 140 CGG GAG TCA CTG AGA ATC TGG AAG AAC ACA GAG AAG GAG AAC GCAACA 600 Arg Glu Ser Leu Arg Ile Trp Lys Asn Thr Glu Lys Glu Asn Ala Thr145 150 155 GTG GCC CAC CTG GTG GGG GCT CTC AGG TCC TGC CAG ATG AAC CTGGTG 648 Val Ala His Leu Val Gly Ala Leu Arg Ser Cys Gln Met Asn Leu Val160 165 170 GCT GAC CTG GTA CAA GAG GTT CAG CAG GCC CGT GAC CTC CAG AACAGG 696 Ala Asp Leu Val Gln Glu Val Gln Gln Ala Arg Asp Leu Gln Asn Arg175 180 185 AGT GGG GCC ATG TCC CCG ATG TCA TGG AAC TCA GAC GCA TCT ACCTCC 744 Ser Gly Ala Met Ser Pro Met Ser Trp Asn Ser Asp Ala Ser Thr Ser190 195 200 205 GAA GCG TCC TGATGGGCCG CTGCTTTGCG CTGGTGGACC ACAGGCATCT793 Glu Ala Ser ACACAGCCTG GACTTTGGTT CTCTCCAGGA AGGTAGCCCA GCACTGTGAAGACCCAGCAG 853 GAAGCCAGGC TGAGTGAGCC ACAGACCACC TGCTTCTGAA CTCAAGCTGCGTTTATTAAT 913 GCCTCTCCCG CACCAGGCCG GGCTTGGGCC CTGCACAGAT ATTTCCATTTCTTCCTCACT 973 ATGACACTGA GCAAGATCTT GTCTCCACTA AATGAGCTCC TGCGGGAGTAGTTGGAAAGT 1033 TGGAACCGTG TCCAGCACAG AAGGAATCTG TGCAGATGAG CAGTCACACTGTTACTCCAC 1093 AGCGGAGGAG ACCAGCTCAG AGGCCCAGGA ATCGGAGCGA AGCAGAGAGGTGGAGAACTG 1153 GGATTTGAAC CCCCGCCATC CTTCACCAGA GCCCATGCTC AACCACTGTGGCGTTCTGCT 1213 GCCCCTGCAG TTGGCAGAAA GGATGTTTTG TCCCATTTCC TTGGAGGCCACCGGGACAGA 1273 CCTGGACACT AGGGTCAGGC GGGGTGCTGT GGTGGGGAGA GGCATGGCTGGGGTGGGGGT 1333 GGGGAGACCT GGTTGGCCGT GGTCCAGCTC TTGGCCCCTG TGTGAGTTGAGTCTCCTCTC 1393 TGAGACTGCT AAGTAGGGGC AGTGATGGTT GCCAGGACGA ATTGAGATAATATCTGTGAG 1453 GTGCTGATGA GTGATTGACA CACAGCACTC TCTAAATCTT CCTTGTGAGGATTATGGGTC 1513 CTGCAATTCT ACAGTTTCTT ACTGTTTTGT ATCAAAATCA CTATCTTTCTGATAACAGAA 1573 TTGCCAAGGC AGCGGGATCT CGTATCTTTA AAAAGCAGTC CTCTTATTCCTAAGGTAATC 1633 CTATTAAAA 1642 208 amino acids amino acid linear protein2 Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser 1 5 1015 Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Gly 20 2530 Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Ser Met 35 4045 Leu Leu Glu Gln Asn Asp Leu Glu Pro Gly His Thr Glu Leu Leu Arg 50 5560 Glu Leu Leu Ala Ser Leu Arg Arg His Asp Leu Leu Arg Arg Val Asp 65 7075 80 Asp Phe Glu Ala Gly Ala Ala Ala Gly Ala Ala Pro Gly Glu Glu Asp 8590 95 Leu Cys Ala Ala Phe Asn Val Ile Cys Asp Asn Val Gly Lys Asp Trp100 105 110 Arg Arg Leu Ala Arg Gln Leu Lys Val Ser Asp Thr Lys Ile AspSer 115 120 125 Ile Glu Asp Arg Tyr Pro Arg Asn Leu Thr Glu Arg Val ArgGlu Ser 130 135 140 Leu Arg Ile Trp Lys Asn Thr Glu Lys Glu Asn Ala ThrVal Ala His 145 150 155 160 Leu Val Gly Ala Leu Arg Ser Cys Gln Met AsnLeu Val Ala Asp Leu 165 170 175 Val Gln Glu Val Gln Gln Ala Arg Asp LeuGln Asn Arg Ser Gly Ala 180 185 190 Met Ser Pro Met Ser Trp Asn Ser AspAla Ser Thr Ser Glu Ala Ser 195 200 205 70 amino acids amino acid singlelinear peptide Modified-site 11 /note= “Val is replaced by Asn for thepoint mutant hFADD” 3 Asp Trp Arg Arg Leu Ala Arg Gln Leu Lys Val SerAsp Thr Lys Ile 1 5 10 15 Asp Ser Ile Glu Asp Arg Tyr Pro Arg Asn LeuThr Glu Arg Val Arg 20 25 30 Glu Ser Leu Arg Ile Trp Lys Asn Thr Glu LysGlu Asn Ala Thr Val 35 40 45 Ala His Leu Val Gly Ala Leu Arg Ser Cys GlnMet Asn Leu Val Ala 50 55 60 Asp Leu Val Gln Glu Val 65 70 70 aminoacids amino acid single linear peptide Modified-site 11 /note= “Ile isreplaced by Asn for the point mutant rFas” 4 Asp Ala Lys Lys Phe Ala ArgGln His Lys Ile Pro Glu Ser Lys Ile 1 5 10 15 Asp Glu Ile Glu His AsnSer Pro Gln Asp Ala Ala Glu Gln Lys Ile 20 25 30 Gln Leu Leu Gln Cys TrpTyr Gln Ser His Gly Lys Thr Gly Ala Cys 35 40 45 Gln Ala Leu Ile Gln GlyLeu Arg Lys Ala Asn Arg Cys Asp Ile Ala 50 55 60 Glu Glu Ile Gln Ala Met65 70 70 amino acids amino acid single linear peptide Modified-site 11/note= “Val is replaced by Asn for the point mutant hFas” 5 Gln Val LysGly Phe Val Arg Lys Asn Gly Val Asn Glu Ala Lys Ile 1 5 10 15 Asp GluIle Lys Asn Asp Asn Val Gln Asp Thr Ala Glu Gln Lys Val 20 25 30 Gln LeuLeu Arg Asn Trp His Gln Leu His Gly Lys Lys Glu Ala Tyr 35 40 45 Asp ThrLeu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys Thr Leu Ala 50 55 60 Glu LysIle Gln Thr Ile 65 70 70 amino acids amino acid single linear peptideModified-site 11 /note= “Leu is replaced by Asn for the point mutanthTNFR-1” 6 Arg Trp Lys Glu Phe Val Arg Arg Leu Gly Leu Ser Asp His GluIle 1 5 10 15 Asp Arg Leu Glu Leu Gln Asn Gly Arg Cys Leu Arg Glu AlaGln Tyr 20 25 30 Ser Met Leu Ala Thr Trp Arg Arg Arg Thr Pro Arg Arg GluAla Thr 35 40 45 Leu Glu Leu Leu Gly Arg Val Leu Arg Asp Met Asp Leu LeuGly Cys 50 55 60 Leu Glu Asp Ile Glu Glu 65 70 9 amino acids amino acidsingle linear 7 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 6 amino acidsamino acid single linear 8 Asp Thr Tyr Arg Tyr Ile 1 5 8 amino acidsamino acid single linear 9 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 117 basepairs nucleic acid single linear 10 AAGCCTGGTA CCATGCTGGG CATCTGGACCCTCCTACCTC TGGTTCTTAC GTCTGTTGCT 60 AGATTATCGT CCAAAGACTA CAAGGACGACGATGACAAGA GTGTTAATGC CCAAGTC 117

What is claimed is:
 1. An isolated nucleic acid molecule comprising asequence encoding a polypeptide comprising amino acid residues 111 to170 of SEQ ID NO:2.
 2. The isolated nucleic acid molecule of claim 1comprising nucleotide 460 to 639 of SEQ ID NO:1.